This invention relates to a photometric apparatus useful in liquid chromatography and, more particularly to such an apparatus which utilizes a sample cell and associated optic means which prevents light rays from impinging on the cell walls or prevents such impinged rays from impinging on downstream detection means.
In a liquid chromatography system, detection of sample constituents is effected by passing the eluent from a liquid chromatography column through a small volume sample cell, passing light through the sample cell and detecting the light emitted from the cell to determine characteristic light absorbance by the sample. It is known that substantial spurious radiation signals are generated by differences in refractive index of the sample and solvents flowing through the cell. These index differences produce complex lens-like effects which can cause a variable proportion of the rays passing through a small photometer cell to impinge on the walls. Since rays which encounter the cell walls are partially absorbed, refractive index variations cause the instrument baseline to be unstable, and sample absorbance values to be in error.
The size and shape of an absorption cell for a high performance liquid chromatograph (HPLC) UV-Visible detector is a compromise between the following three factors. First, it is important to pass a high light flux through the cell to achieve a high signal-to-noise-ratio measurement. Second, the cell volume must be kept low to prevent peak spreading and loss of chromatographic resolution. Third, for a given cell volume and optical throughput, the cell pathlength should be as long as possible to maximize sample absorption. These factors lead to the typical dimensions of a conventional HPLC-UV-Vis absorption flow cell; 10 mm long, 1 mm diameter and about 8 micro liters volume. In processes wherein samples are caused to fluoresce and the amount of fluorescence is measured, light rays striking the cell walls also is a problem. This is due to the fact that there is some adsorbance of previously analyzed samples by the transparent cell walls which fluoresces when exposed to light rays and thereby causes spurious signals.
It has been proposed in U.S. Pat. Nos. 4,011,451 and 4,276,475 to provide a flow-cell whereby the lens effect is rapidly dissipated by a progressive increase in the cross-sectional area of the flow-cell along the flow-path. The wall of the flow-cell forms a diverging surface of rotation whereby the walls form an angle of divergence of several degrees with the axis of the cell. The optical system avoids any substantial radiation from entering the cell at sharp angles which could result in the radiation impinging on the walls of the cell. The divergence of the cell walls substantially dissipates the undesirable effect of the refractive index gradients encountered in HPLC separations. Angles of divergence between the axis of the flowpath and the wall of the cell of 1.degree. to 3.degree. are disclosed to be most advantageous.
While the tapered or conically shaped cells described in these two patents provides substantial advantages over the prior art, they have some disadvantages which would desirably be overcome. The required cell volume is undesirably increased as compared to a cylindrically shaped cell. Increased sample cell volume causes band spreading of the light passing through the sample cell. In addition, a conically shaped cell is more difficult to produce and therefore is more expensive than a cylindrically shaped cell having a constant circular cross-section. Therefore, it would be desirable to provide a means to effect liquid chromatography which permits minimizing sample cell volume and which avoids the need for a non-cylindrically shaped sample cell, while still preserving high optical throughput and insensitivity to refractive index changes.